Fig 2: ASH1L is required for gene expression regulation in the cell cycle and apoptosis. (A) Western blot analysis of ASH1L protein levels in different cancer cell lines. The GAPDH was used as a loading control. (B) Western blot analysis of H3K4me2/3 and H3K36me2 levels after Flag-ASH1L-C or Flag-ASH1L-C-ΔPHD overexpressed in 293T cells. (C) The knockdown efficiency of siRNAs targeting ASH1L (siASH1L) was detected by qRT-PCR at 48 h after siNC and siASH1L were transfected to PC3 or DU145 prostate cancer cell lines. (D) The analysis of differentially expressed genes (DEGs) in the cellular processes branch of the KEGG pathway. The RNA-seq result is from the three replicates of the control (siNC) group and ASH1L knockdown (siASH1L#1) group PC3 cells. (E) GSEA of RNA-seq data to analyze the signaling pathways altered by ASH1L knockdown in PC3 cells. (F) Heatmap showing changes in the expression of indicated genes in the control (siNC) and ASH1L knockdown (siASH1L#1) PC3 cells. (G) qRT-PCR analysis of cell cycle–related gene expression in the control (siNC) and ASH1L knockdown (siASH1L#1) PC3 cells. (H) Western blot analysis of cell cycle and apoptosis pathway–related proteins in the control (siNC) and ASH1L knockdown (siASH1L#1) PC3 cells. The GAPDH was used as loading control. The density of every band was quantified using Image J software. (These experiments were repeated at least three times, and the error bars represent at least three replicates. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig 3: ASH1L knockdown induced cell cycle arrest and apoptosis but did not affect cell viability. (A) Representative flow cytometry images about the cell cycle distribution of PC3 at 48 h after siNC and siASH1L#1 transfection. (B) Cell cycle analysis of PC3 at 24, 48, and 72 h after siNC and siASH1L#1 transfection. (C) Representative flow cytometry images showing the proportion of early apoptotic (Annexin V+ PI-) and late apoptotic (Annexin V+ PI+) PC3 cells at 48 h after siNC and siASH1L#1 transfection. (D) Apoptosis level of PC3 detected by Annexin V/PI staining at 24, 48, and 72 h after siNC and siASH1L#1 transfection. (E) Representative flow cytometry images showing the proportion of early apoptotic (Annexin V+ PI-) and late apoptotic (Annexin V+ PI+) DU145 cells at 48 h after siNC and siASH1L#1 transfection. (F) Apoptosis level of DU145 detected by Annexin V/PI staining at 24, 48, and 72 h after siNC and siASH1L#1 transfection. (G) Plate colony formation was performed in PC3 cells transfected with siNC and siASH1L#1 and incubated for 9 days. Representative colony images are shown. (H) Analysis of cell viability by the MTT assay in siNC- and siASH1L#1-transfected PC3 cells. The plates were detected at 72 h after siNC and siASH1L#1 were transfected. (These experiments were repeated at least three times, and the error bars represent at least three replicates. *, p < 0.05, **, p < 0.01, ****, p < 0.0001).

Fig 4: Structure of ASH1L PHD finger binding with the H3 (1–15) K4me2 peptide. (A) Cartoon representation of the NMR structure of the ASH1L PHD finger (light blue)/H3K4me2 peptide (yellow). (B) Electrostatic potential surface representation of the ASH1L PHD finger binding with H3K4me2 peptide in stick depiction (color-coded by atom type). The amino acid around the K4 residue and in the β2 strand are labeled. (C) The key residues involved in the intermolecular interactions at the protein/peptide interface are labeled. These side chains are color-coded by the atom type and showed in the stick model. (D) The key side chains of binding groove around K4me2 are picked out.

Fig 5: ASH1L PHD finger shows comparable binding affinity to H3K4me1/me2/me3. (A) Schematic representation of ASH1L protein. (B) Histone peptide array probed with the His-tagged ASH1L PHD finger. (C) Binding affinity of ASH1L PHD for the indicated H3 (1–15) peptides measured by isothermal titration calorimetry (ITC) and NMR titration. (D) Representative binding carves used to determine the KD values by ITC. (E) Superimposed ¹H,15N HSQC spectra of the ASH1L PHD finger collected upon titration with the indicated peptides in a protein–peptide molar ratio of 1: 5. Spectra are color-coded according to different peptides. Arrows indicate chemical shift changes. (F) Representative saturation curves used to determine the KD values by NMR titration. The saturation curves were calculated using a single-site binding equilibrium model.